Abstract

The complete genome sequence of the crenarchaeon Ignicoccus hospitalis published recently in Genome Biology provides a great leap forward in the dissection of its unique association with another
hyperthermophilic archaeon, Nanoarchaeum equitans.

A unique association in the archaeal world

The discovery of Nanoarchaeum equitans in a hydrothermal vent off the coast of Iceland by the group of Karl Stetter in 2002
has been one of the most exciting findings of the past decade in microbiology [1,2]. This tiny regular coccus (400 nm = 1% of the volume of Escherichia coli) lives at the surface (equitans meaning 'riding') of its host, the crenarchaeon Ignicoccus hospitalis (hospitalis referring to the ability to serve as a host for N. equitans), which belongs to the order Desulfurococcales within the Crenarchaeota (Figure 1). Members of the genus Ignicoccus are the only obligatory anaerobic chemolithoautotrophic sulfur reducers within the
Desulfurococcales, coupling elemental sulfur respiration and carbon dioxide fixation
through a novel and unique pathway called the dicarboxylate/4-hydroxybutyrate cycle
and using molecular hydrogen as electron donor ([3] and references therein).

Figure 1.(a) Schematic diagram of archaeal phylogeny showing the evolutionary relationships between
archaeal lineages for which completely sequenced genomes are available (updated from
[12]). The positions of Ignicoccus hospitalis and Nanoarchaeum equitans are indicated by arrows. The position of N. equitans has been controversial [2,8], but the position suggested here has been recently supported by a specific synapomorphy
(a derived character state shared by two or more groups) [13] and signature genes [14]. (b) Electron micrograph showing a cell of N. equitans attached to a cell of I. hospitalis. The scale bar is 100 nm. Courtesy of Dr Rachel Reinhard, Regensburg University.
OM: outer membrane.

Ignicoccus species exhibit irregular coccus morphology and are the only known archaeal cells
to be surrounded by two membranes ([3,4] and references therein). The cytoplasmic membrane is separated from an outer membrane,
which has a distinct lipid composition and contains pores of unique type, by a periplasmic
space with a variable width of between 20 and 500 nm. The association between N. equitans and I. hospitalis is particularly interesting because it is the first (and so far the only) known example
of a parasitic/symbiotic partnership involving two archaea, and moreover two hyperthermophilic
organisms [1]. N. equitans cannot be cultivated in the absence of I. hospitalis, whereas the latter thrives well without its putative symbiont [1]. Although no benefits for the host can be detected in co-cultures, N. equitans and I. hospitalis are pioneering colonizers in deep-sea hydrothermal vents [5], and their association has been proposed to help these chemolithotrophs compete with
heterotrophs in sulfide-rich environments. Often, the genome of only one of the two
partners in a symbiotic association has been sequenced. That of N. equitans has already been sequenced [2] and, fortunately, the genome of I. hospitalis has now been published in Genome Biology by Podar et al. [3], allowing for the first time a comprehensive genomic analysis of this unique archaeal
association.

A host with a highly reduced genome

The most important finding of Podar et al. is that of few but striking similarities between the genomes of I. hospitalis and N. equitans. First, both have highly reduced genomes: N. equitans harbors a compact genome of 490 kb encoding 552 genes [2], which represents the smallest known genome for an exosymbiont, whereas the genome
of I. hospitalis is around 1.3 Mb, the smallest among free-living organisms [3]. As pointed out by Podar et al., even the combined gene complement of both genomes is significantly smaller than
that of the average free-living bacteria or archaea [3]. The authors put forward the hypothesis that, as with N. equitans [6,7], the small size of the genome of I. hospitalis is a derived trait, resulting from a massive reduction that occurred after the divergence
of this crenarchaeon from other members of the Desulfurococcales [3]. They estimate that around 500 genes corresponding to archaeal-specific clusters
of orthologous genes (arCOGs) [7], including 19 genes otherwise common to all crenarchaeal genomes, have been lost
in the I. hospitalis genome [3]. The authors put forward the hypothesis that these losses may be linked to the adaptation
of Ignicoccus to a strict anaerobic and autotrophic lifestyle compared with its relatives.

Moreover, as in the case of N. equitans, the genome of I. hospitalis shows little operon-structure conservation, very few paralogs and no transposable
elements, indicating that both genomes have been streamlined by extensive chromosomal
rearrangements. This process has gone further in the smaller N. equitans genome [2], and it has been proposed that these features are primitive, leading to the hypothesis
that this archaeon is a living fossil [8]. To us, the fact that N. equitans and I. hospitalis have these same features seems to suggest instead that both genomes evolved through
a similar reduction process, in agreement with the position of N. equitans in archaeal phylogenies as an euryarchaeon possibly related to the Thermococcales
[6] (Figure 1).

Expanded gene families

Interestingly, despite an evolutionary trend toward reduction, the genome of I. hospitalis exhibits expansions of specific gene families, notably those coding for proteins harboring
domains involved in the formation of macromolecular complexes, such as WD40 repeats,
CBS and V4R domains [3]. Moreover, the genome of I. hospitalis has the lowest arCOG coverage among the Crenarchaeota: an important fraction of its
open reading frames (approximately 20%) cannot be affiliated to any arCOG [3].

The experimental characterization of these novel proteins will surely help to decipher
at the molecular level the unique mechanisms responsible for the association between
I. hospitalis and N. equitans. The expanded family of proteins with V4R domains will be especially interesting
to study, because these proteins are homologs to the Bet3 subunit of the TRAPPI vesicle-tethering
complex that is conserved in all eukaryotes [9]. Microscopy observations have consistently shown that both round and elongated membrane-coated
vesicles are released from the cytoplasmic membrane in the periplasm of I. hospitalis and sometimes appear to come into close proximity to, and fuse with, the outer membrane
([4] and references therein). This has led to the hypothesis that these vesicles might
be involved in transport of metabolites from the host to N. equitans. It will be important to determine whether proteins with VR4 domains indeed participate
in the vesicle-trafficking system observed in I. hospitalis and if these vesicules are really involved in the interaction with N. equitans. Alternative hypotheses suggest that transport of metabolites or substrates between
the two cells occurs through unusual structures observed at the contact point between
them ([10] and references therein). These structures could involve some of the I. hospitalis-specific proteins revealed by the genome analysis.

Horizontal gene transfer in a minimalist system

Using phylogenetic methods, Podar et al. [3] have identified a number of genes that were probably acquired by horizontal gene
transfer (HGT), either from bacteria (4%) or from Euryarchaeota (6%). Although limited
in extent, some of these HGT events might have been important in permitting the combination
of a streamlined genome with efficient metabolic strategies. For instance, I. hospitalis may use a transporter acquired from a euryarchaeon to import ammonium for nitrogen
assimilation, a wise strategy in a highly reduced environment [9]. Similarly, I. hospitalis may use a sulfur/polysulfide reductase complex of bacterial origin in addition to
the one normally found in Crenarchaeota [3].

Podar et al. [3] present an extensive description of I. hospitalis genes involved in genetic information processing, transport, central metabolism, respiration
and energy metabolism. A major question is whether some of the genes for metabolites
and energy production originally present in the N. equitans genome were transferred to the genome of I. hospitalis and the gene products are now being imported back into N. equitans. HGT from N. equitans to I. hospitalis and vice versa has apparently occurred, but on a very limited scale (only 13 such genes were identified),
indicating that N. equitans should be able to import and use metabolites and energy (ATP) from I. hospitalis, as previously experimentally demonstrated in the case of lipids and amino acids
[10].

What next?

The discovery of the unique N. equitans/I. hospitalis system has significantly increased our knowledge of the archaeal domain in terms of
its diversity (the discovery of a new main lineage), ecology (association between
two archaea) and genomics (highly reduced archaeal genomes). The sequencing of the
genomes of both partners now provides valuable data for elucidating the nature of
this association. Important biological questions remain to be answered, however: for
instance, we would like to know how the cell cycles of N. equitans and I. hospitalis are coordinated. Another important question concerns the wide diversity of associations
involving a nanoarchaeal partner. Up to now, the association between I. hospitalis and N. equitans has been described as highly specific because attempts to infect other species of
Ignicoccus or other hyperthermophilic archaea with N. equitans have failed. However, in the past few years, molecular ecological studies have identified
nanoarchaeal sequences in hot marine and terrestrial environments from geographically
distant regions, suggesting that nanoarchaeota are widely distributed around the world.
More surprisingly, nanoarchaeota have recently been reported from hypersaline mesophilic
environments [11]. It will be extremely interesting to determine if these sequences correspond to free-living
nanoarchaea or if they are symbiotic/parasitic cells that have established associations
similar to that between N. equitans and I. hospitalis. In the latter case, the presence of nanoarchaea in halophilic and mesophilic environments
might involve hosts other than I. hospitalis or Desulfurococcales, because no mesophiles are currently known in these lineages.
The characterization of these uncultivated nanoarchaea as well as that of their hosts
will surely bring answers to these questions.

Acknowledgements

We thank Dr Rachel Reinhard for useful comments and the micrograph in Figure 1b.